Original Article Clinical Implication of Long Non-Coding RNA NEAT1 Expression in Hepatocellular Carcinoma Patients

Int J Clin Exp Pathol 2015;8(5):5395-5402 www.ijcep.com /ISSN:1936-2625/IJCEP0006987

Original Article Clinical implication of long non-coding RNA NEAT1 expression in hepatocellular carcinoma patients

Sien Guo1*, Wenjie Chen2*, Yihuan Luo2, Fanghui Ren2, Tengfei Zhong2, Minhua Rong3, Yiwu Dang2, Zhenbo Feng2, Gang Chen2

1Department of Hepatobiliary Surgery, First Affiliated Hospital of Guangxi Medical University, 6 Shuangyong Road, Nanning, Guangxi Zhuang Autonomous Region 530021, P. R. China; 2Department of Pathology, First Affiliated Hospital of Guangxi Medical University, 6 Shuangyong Road, Nanning, Guangxi Zhuang Autonomous Region 530021, P. R. China; 3Department of Research, Affiliated Cancer Hospital, Guangxi Medical University, 71 Hedi Road, Nanning, Guangxi Zhuang Autonomous Region 530021, P. R. China. *Equal contributors. Received February 11, 2015; Accepted April 12, 2015; Epub May 1, 2015; Published May 15, 2015

Abstract: Hepatocellular carcinoma (HCC), a primary malignancy of the liver, is associated with high mortality rate and poor prognosis. Emerging evidence showed that novel biomarkers are required toward a better understanding of the biological mechanisms of HCC. NEAT1 (nuclear paraspeckle assembly transcript 1, also known as MENε/β), a novel long non-coding RNA (lncRNA), serves as a crucial regulator in several cancers. However, the correlation between NEAT1 expression with tumorigenesis and metastasis in HCC tissues remains out of the question so far. In the current study, the aim was to evaluate the potential role of NEAT1 expression in HCC tissues and its relationship with clinicopathological parameters. Method: The expression of NEAT1 was detected by qRT-PCR, in 95 cases of adjacent non-cancerous liver and their paired HCC tissues, respectively. The associations of NEAT1 with clinicopathological features and other biological factors were further analyzed. Result: Our results revealed that NEAT1 appeared to have higher expression in the HCC tissues, compared with the adjacent non-cancerous liver tissues. High levels of NEAT1 promoted the clinical features of HCC, including the number of tumor nodes, metastasis, clinical TNM stage, the status of portal vein tumor embolus, vaso-invasion and the infiltration of tumor cells. Ad- ditionally, high NEAT1 expression levels were significantly associated with the expression level of MDTH, NM23 and MALAT1. Conclusion: Our study demonstrates that NEAT1 acts as a pivotal player in tumorigenesis and metastasis of hepatocellular carcinoma.

Keywords: NEAT1, hepatocellular carcinoma, tumorigenesis, metastasis

Introduction Recent evidences have pointed to a relationship between several long non-coding RNAs Liver cancer ranks the fifth among all cancers (lncRNAs) and metastasis, drug resistance and worldwide and it is the third most frequent etio- other clinical outcome in several types of can- logical factor of cancer-caused deaths. Hepato- cers [8-13]. Essentially, lncRNAs are involved in cellular carcinoma (HCC), the predominant the pathogenesis of HCC, such as HOX tran- form of adult liver malignancies, accounts for script antisense RNA (HOTAIR), metastasis between 85% and 90% of liver cancers [1-3]. Several risk factors are related to the tumori- associated lung adenocarcinoma transcript 1 genesis of HCC, including chronic hepatitis (MALAT1) and H19 [14-17]. NEAT1, a nuclear- virus infection and fatty liver diseases [4-7]. restricted long non-coding RNA, encodes two Previous studies have not proven adequate evi- isoforms: 3700-nucleotide (nt) NEAT1_1 and dences for the prevention, diagnosis and treat- 23,000-nt NEAT1_2 [18]. This non-coding RNA ment of HCC. Thus, understanding and insight has been recently revealed to localize specifi- into the mechanisms contributing to tumorigen- cally to paraspeckles. And NEAT1 is involved in esis and aggressiveness in HCC is necessary the process of gene expression regulation by and essential for more precise diagnosis and retaining mRNAs for editing in the nucleus [19]. effective treatments. Cumulatively, studies suggest that down-regu- NEAT1 in hepatocellular carcinoma lated NEAT1 expression is involved in lung, liver, using Applied Biosystems PCR7900. The expre- esophageal and retinal cancers [20]. Intere- ssion of NEAT1 was examined by Reverse tran- stingly, evidence also argued that high NEAT1 scription (RT) and qPCR kits according to the expression is involved in the metastasis of can- instructions described previously [22, 23]. Pri- cers like lung cancer [21]. To date, the emerg- mers were as below: NEAT1 Forward-5’-TGGC- ing potential role of NEAT1 in HCC is yet unclear. TAGCTCAGGGCTTCAG-3’, NEAT1 Reverse-5’-TC- TCCTTGCCAAGCTTCCTTC-3’; GAPDH Forward- In light of these findings and previous reports, 5’-TGAACGGGAAGCTCACTGG-3’, GAPDH Rever- we hypothesized an appealing association be- se-5’-TCCACCACCCTGTTGCTGTA-3’. The NEAT1 tween NEAT1 expression and HCC. To investi- expression was calculated with the formula gate this hypothesis, we evaluated the expres- 2-Δcq [22-24]. sion of NEAT1 in adjacent non-cancerous liver and HCC tissues, in a set of 95 primary tumors, Statistics and investigated the relationship between NEAT1 expression and clinical, histological, All statistical analyses were performed using pathological and other biological factors in SPSS 20. For the analysis of the significance of HCC. difference between two groups, we performed Student’s t test. Accordingly, we also applied Materials and methods One-way analysis of variance (ANOVA) test to distinguish its significance of difference, as we Patients divided differentiation into three groups. The strengths of the associations between NEAT1 Patients were selected from the First Affiliated expression and clinicopathological parameters Hospital of the Guangxi Medical University were tested with the Spearman rank correla- (Nanning, Guangxi, China) during March, 2010 tion. The effectiveness of NEAT1 for predicting to December, 2011. Seventy-five males and the risk of the clinicopathological parameters twenty female individuals were enrolled in the was evaluated by ROC curve. We used Kaplan- study, with ages between 29 and 82 years old. Meier survival method and log-rank test to esti- Additionally, the clinicopathological parameters mate the impact of NEAT1 on recurrence of were collected such as age, gender, differentia- HCC. All tests were 2-sided, and statistical sig- tion, tumor size, tumor nodes, metastasis, clini- nificance was considered as P<0.05. cal TNM stages, the presence or absence of portal vein tumor embolus, vaso-invasion, cap- Results sular infiltration and cirrhosis, and other biomarkers, for instance, serum AFP level detect- Overexpression of NEAT1 in HCC ed by ELISA; expression of metadherin (MTDH), NM23, P53, P21, vascular endothelial growth Analysis of 95 matched samples (95 adjacent factor (VEGF), as well as microvessel density non-cancerous liver tissues and 95 HCC tis- (MVD) stained by using CD34 with immunohis- sues) by RT-qPCR detected significantly tochemistry; and expression of a lncRNA, increased levels of NEAT1 in tumor samples MALAT1 by using real time RT-qPCR. Table 1 compared to the non-tumor group (6.42±3.55 showed all these features in detail. The Ethical vs. 5.29±3.14, P=0.02, Figure 1A). Committee of First Affiliated Hospital, Guangxi Medical University, China approved the current Associations of NEAT1 with clinicopathological study. We obtained informed consent from all factors participating patients. In accordance with the Subsequently, we explored whether there was Helsinki Declaration, related research proce- dure was carried out. All samples were reviewed an association of NEAT1 expression levels with and diagnosed by two independent patho- well-established biological factors in 95 HCC logists. tissues. Correlations of NEAT1 expression of HCC and tumor characteristics were shown in RT-qPCR Table 1. Most notable findings were the strong associations between high HCC expression lev- RNA isolation and RNA normalization were per- els and some clinicopathological parameters, formed by the method reported before [22, 23]. such as, multi-tumor nodes (r=0.223, P=0.030), Extracted RNA was analyzed by Real-time qPCR metastasis (r=0.398, P<0.001), portal vein

5396 Int J Clin Exp Pathol 2015;8(5):5395-5402 NEAT1 in hepatocellular carcinoma

Table 1. Association of NEAT1 expression with clinicopathological parameters in HCC Neat1 relevant expression (2-ΔCq) Clinicopathological Feature n Mean ± SD t P Tissue Adjacent non-cancerous liver 95 5.2859±3.14222 2.34 0.02 HCC 95 6.4237±3.54655 Age ≥50 46 6.5646±3.62168 0.373 0.71 <50 49 6.2914±3.50690 Gender male 75 6.5925±3.61357 0.898 0.372 female 20 5.7905±3.29189 Differentiation high 6 4.1500±1.67660 F=1.390a 0.254 moderate 60 6.6692±3.81677 low 29 6.3862±3.12883 Size <5 cm 18 6.3317±3.55015 0.122 0.903 ≥5 cm 77 6.4452±3.56866 Tumor nodes single 52 5.6040±2.83613 -2.675 0.009 multi 43 7.4149±4.06767 Metastasis Without metastasis 46 4.8935±2.13607 -4.546 <0.001 With metastasis 49 7.8602±4.00047 Clinical TNM stage I~II 22 5.2659±2.47973 -2.192 0.033 III~IV 73 6.7726±3.75431 Portal vein tumo embolus - 63 5.7516±3.17703 -2.465 0.016 + 32 7.7469±3.90225 Vaso-invasion - 59 5.3249±2.47388 -3.709 0.001 + 36 8.2244±4.27418 Tumor capsular infiltration With complete capsule 45 5.0787±2.23320 -3.849 <0.001 No capsule or infiltration 50 7.6342±4.06132 HCV - 63 6.3737±3.59428 -0.192 0.848 + 32 6.5222±3.50529 HBV - 17 6.9465±3.86748 0.669 0.505 + 78 6.3097±3.48905 AFP - 41 6.2549±3.38778 0.47 0.64 + 38 6.6421±3.92849 Cirrhosis - 50 6.1410±3.13986 0.817 0.416 + 45 6.7378±3.96238 NM23 - 20 4.5125±2.25753 -3.676 0.001 + 75 6.9333±3.66313 MTDH - 50 5.3994±2.88525 -3.978 <0.001 + 39 8.2485±3.67527 P53 - 40 6.2882±3.28013 -0.316 0.753 + 55 6.5222±3.75514 P21 - 62 6.4311±3.25098 0.028 0.978 + 33 6.4097±4.09887 VEGF - 25 5.5120±2.99639 -1.507 0.135 + 70 6.7493±3.68832 Ki-67 LI Low 47 5.7415±3.11216 -1.88 0.063 High 48 7.0917±3.84129 MVD Low 47 6.3757±3.75190 -0.13 0.897 High 48 6.4706±3.37257 MALAT1 Low 52 5.3667±2.63275 -3.235 0.002 High 43 7.7019±4.08391 aANOVA.

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Figure 1. A. Overexpression of NEAT1 in HCC. Higher NEAT1 expression level was observed in HCC compared to that in the adjacent non-cancerous live (P=0.020). B. The relationships between NEAT1 clinicopathological features. Nodes: 1. multi-tumor nodes; 2. single tumor node; Metastasis: 1. with metastasis; 2. without metastasis; TNM: 1. TNM III-IV 2. TNM I-II; Embolus: 1. portal vein tumor embolus (+); 2. portal vein tumor embolus (-); Vaso-invasion: 1. (+); 2. (-); Capsular: 1. no capsule or infiltration; 2. With complete capsule; C. Associations of NEAT1 expression with biological factors in HCC. NM23:1. (+); 2. (-); MDTH: 1. (+); 2. low level (-); MALAT1: 1. high expression 2. low expression; *P<0.05; **P<0.01; ***P<0.001.

P=0.009). NEAT1 level in HCC tissues with metastasis was remarkably upregulated (7.86±4.00) as compared to that without metastasis (4.89±2.14, P<0.001). Significantly increasing level of NEAT1 was found in clinical TNM stage III~IV (6.77±3.75), as compared to that in the stage I~II (5.27±2.48, P=0.033). NEAT1 appeared to have higher expression in the HCC tissues with the portal vein tumor embolus (7.75±3.90) than that without (5.75±3.18, P=0.016). Moreover, higher NEAT1 expression was detected in vaso-invasion (8.22±4.27) than that without (5.32±2.47, Figure 2. The Kaplan-Meier survival curve for the im- P=0.001). Furthermore, NEAT1 levels were also plication of NEAT1 expression in HCC patients. significantly higher in the group of tumors without capsule or with tumor cell infiltration (7.63±4.06) than that in the corresponding tumor embolus (r=0.255, P=0.013), vaso-inva- group (5.08±2.23, P<0.001, Figure 1B). sion (r=0.341, P=0.001), tumor capsule infiltra- Additionally, the level of NEAT1 was significantly tion (r=0.324, P=0.001), MTDH level (r=0.278, higher in high MTDH group (8.25±3.68) than in P<0.001), NM23 level (r=0.287, P=0.005) and low MDTH group (5.40±2.89, P<0.001). The MALAT1 level (r=0.180, P=0.013). Meanwhile, positive of NM23 also disclosed a higher NEAT1 level in HCC with multi-tumor nodes was expression of NEAT1 (6.93±3.66) than the neg- significantly higher (7.41±4.07) than that in the ative one (4.51±2.26, P=0.001). Additionally, group with single tumor node (5.60±2.84, the high MALAT1 expression group revealed a

5398 Int J Clin Exp Pathol 2015;8(5):5395-5402 NEAT1 in hepatocellular carcinoma

Figure 3. A. The significant diagnostic value of NEAT1 for HCC (AUC=0.594, 95% CI: 51.29%-67.50%, P=0.025). B. The predicative value NEAT1 for capsule or infiltration resulted in AUC of 0.687 (95% CI: 57.85%-79.53,P =0.02); C. NEAT1 was considered to be a valuable parameter in the prediction of tumor node (AUC=0.629, 95% CI: 51.41%- 74.44%, P=0.031); D. The AUC was 0.73 for metastasis (95% CI: 51.29%-67.5%, P<0.001); E. The AUC was 0.656 for the portal vein tumor embolus (95% CI: 53.33%-77.8%, P=0.014); F. The AUC for vaso-invasion was 0.703 (95% CI: 58.65%-81.89%, P=0.001). significantly higher NEAT1 level (7.70±4.08) an=5.90). The mean recurrence time was than the low expression group (5.37±2.63, 58.90 months in patients with higher NEAT1 P=0.002, Figure 1C). We next divided the expression and 50.60 months in patients with patients into two groups using the median lower NEAT1 expression, but there is no statisti- NEAT1 expression as cutoff value (medical significance P( =0.579, Figure 2).

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Diagnostic value of NEAT1 for clinicopathologi- one to indicate a direct association of NEAT1 cal parameters by ROC curve analysis expression with liver cancers via GEO acces- sioned human short SAGE libraries. Inter- As the ROC curve indicated, NEAT1 of 3.7 repre- estingly, NEAT1 presented a lower expression sented a significant diagnostic cut off value for in liver cancers than in normal tissues in the HCC (AUC=0.594, P=0.025, Figure 3A). We fur- previous study [20]. Contrary to the result of ther explored the predictive value of NEAT1 for Gibb et al., our result revealed that high NEAT1 other clinicopathological parameters in the 95 expression was detected in HCC. A possible HCC samples by ROC curve. NEAT1 of 6.32 in explanation for this discrepancy is that NEAT1 the prediction of capsule or infiltration resulted was detected both in human and non-human in AUC of 0.687 (P=0.02, Figure 3B). As NEAT1 origin in their study. In our current study, the ≥6.45, it was also considered to be a valuable significant higher expression of NEAT1 was parameter in the prediction of tumor node detected in 95 cases of HCC tissues, compared (AUC=0.629, P=0.031), metastasis (AUC=0.73, with adjacent non-cancerous liver tissues. P<0.001), the portal vein tumor embolus Taken together with the previous studies, we (AUC=0.656, P=0.014) and vaso-invasion demonstrate that the regulation of NEAT1 con- (AUC=0.703, P=0.001), respectively (Figure tributes to tumorigenesis in HCC. Further large- 3C-F). scale studies might facilitate the discovery of NEAT1 molecular regulation in hepatocarcino- Discussion genesis of HCC.

Accumulating evidence indicates that the It was interesting to discover the correlation between NEAT1 expression level and clinico- involvement in functional lncRNAs is implicated pathological parameters in HCC. The expres- in HCC pathogenesis [25]. For example, GAS5 sion level of NEAT1 was strongly correlated with expression was decreased in human HCC and the numbers of tumor nodes, metastasis and was associated with advanced tumor progres- TNM stage in HCC patients. Moreover, we found sion [26]. Moreover, MALAT1 expression level significant correlations between high NEAT1 was proved to be an independent prognostic levels and portal vein tumor embolus, vaso- factor for HCC. Furthermore, the higher expres- invasion and tumor capsular infiltration. The sion of MALAT1 was also found to be related results also indicate that NEAT1 affects the with shortened disease‑free survival post liver infiltration of tumor cells, migration and inva- transplantation [16]. Another lncRNA overex- sion in HCC. More specifically, NEAT1 appears pressed in HCC was HOTAIR. HCC patients with to be associated with MTDH level. The previous high expression of HOTAIR exhibited significant- studies suggested that MTDH was regarded as ly poorer prognosis than those with low HOTAIR a major event in metastasis and poor outcome expression [15]. in HCC patients [31, 32]. Of note, higher NEAT1 expression was detected in the cases of NM23 NEAT1 has been extensively demonstrated to being positive. RUN et al carried out the study be involved in several cancers. Kim et al. have that overexpression of NM23 contributes to the demonstrated that NEAT1 was up-regulated in hepatocarcinogenesis in HCC [33]. Given the stage III serous ovarian carcinoma [27]. A strong association with NM23 level, the evi- recent study described that upregulation of dence of upregulated NEAT1 expression in HCC NEAT1 was observed in acute promyelocytic suggests a novel pro-oncogenic potential role leukemia samples and cell lines [28]. Dimple et in hepatocarcinogenesis. All together, our data al. conducted the study that NEAT1 could act as indicate that the upregulation of NEAT1 increas- one of the differentially regulated lncRNAs in es tumorigenesis and metastasis in HCC. prostate cancer by activating prostate cancer genes [29]. The report by Choudhry et al. also The biological mechanisms of NEAT1 regulation showed that high tumor NEAT1 expression con- in HCC remain currently poorly understood. In fers a poor outcome in breast cancer patients prostate cancer, NEAT1 is involved in modula- [30]. The objective of this study was to seek to tion of oncogenic growth by altering the epigen- probe the potentials of NEAT1, a long non-cod- etic landscape [29]. Inducted by hypoxia in HIF ing RNA recently characterized by our group in transcriptional pathways, NEAT1 also plays an HCC. To our knowledge, Gibb et al. was the first important role in promoting proliferation and

5400 Int J Clin Exp Pathol 2015;8(5):5395-5402 NEAT1 in hepatocellular carcinoma reducing apoptosis, contributing to tumorigen- nomous Region 530021, P. R. China. Fax: +86 771 esis [30]. More recently, the cellular levels of 5358943; E-mail: [email protected] (GC); NEAT1_1/2 were found to be down-regulated in [email protected] (ZBF) a particular class of cells in MALAT1-knockout References mice, which indicates that MALAT1 transcribed from the adjacent region has an effect on the [1] Ma L, Chua MS, Andrisani O and So S. Epi- expression of NEAT1_1/2 in specific cells [34]. genetics in hepatocellular carcinoma: an up- In agreement with the previous report, high date and future therapy perspectives. World J NEAT1 expression was found to be associated Gastroenterol 2014; 20: 333-45. [2] Lafaro KJ, Demirjian AN and Pawlik TM. Epide- with MALAT1 upregulation in our study. MALAT1 miology of Hepatocellular Carcinoma. Surg On- has been associated with metastasis and poor col Clin N Am 2015; 24: 1-17. prognosis of HCC according to histology find- [3] Taura N, Fukushima N, Yastuhashi H, Takami Y, ings. MALAT1 is well-known as an oncogene, Seike M, Watanabe H, Mizuta T, Sasaki Y, Na- because it regulates alternative splicing of gata K, Tabara A, Komorizono Y, Taketomi A, endogenous target genes that are involved in Matsumoto S, Tamai T, Muro T, Nakao K, Fu- cancer [16]. Moreover, we hypothesized that kuizumi K, Maeshiro T, Inoue O and Sata M. The incidence of hepatocellular carcinoma as- NEAT1 may play an essential role in the prog- sociated with hepatitis C infection decreased ress and metastasis of HCC, by affecting the in Kyushu area. Med Sci Monit 2011; 17: Ph7- expression of MALAT1. Taken together, the 11. results suggest NEAT1 may modulate in tumori- [4] El-Serag HB and Rudolph KL. Hepatocellular genesis and metastasis in HCC. The molecular carcinoma: epidemiology and molecular carci- mechanism driving this regulation is still nogenesis. Gastroenterology 2007; 132: unclear. 2557-76. [5] Hou W and Bonkovsky HL. Non-coding RNAs in In conclusion, our results suggest that NEAT1 hepatitis C-induced hepatocellular carcinoma: dysregulation and implications for early detec- may represent a valuable predictive marker of tion, diagnosis and therapy. World J Gastroen- tumorigenesis and metastasis of human HCC. terol 2013; 19: 7836-45. Additional research of larger series into the [6] Kretzer IF, do Livramento A, da Cunha J, Gon- molecular mechanism of NEAT1 in HCC is calves S, Tosin I, Spada C and Treitinger A. needed. Hepatitis C worldwide and in Brazil: silent epi- demic--data on disease including incidence, Acknowledgements transmission, prevention, and treatment. Sci- entificWorldJournal 2014; 2014: 827849. The study was supported partly by the Fund of [7] Amr S, Iarocci EA, Nasr GR, Saleh D, Blancato Guangxi Natural Scientific Research (No. J, Shetty K and Loffredo CA. Multiple pregnan- 2013GXNSFBA019191), Guangxi Provincial cies, hepatitis C, and risk for hepatocellular Health Bureau Scientific Research Project carcinoma in Egyptian women. BMC Cancer 2014; 14: 893. (Z2014054), Youth Science Foundation of [8] Zhang H, Chen Z, Wang X, Huang Z, He Z and Guangxi Medical University (GXMUYSF201311), Chen Y. Long non-coding RNA: a new player in Guangxi University Science and Technology cancer. J Hematol Oncol 2013; 6: 37. Research Projects (LX2014075), and the Fund [9] Malek E, Jagannathan S and Driscoll JJ. Corre- of National Natural Science Foundation of lation of long non-coding RNA expression with China (NSFC 81360327). The funders had no metastasis, drug resistance and clinical out- role in study design, data collection and analy- come in cancer. Oncotarget 2014; 5: 8027-38. [10] Mizrahi I, Mazeh H, Grinbaum R, Beglaibter N, sis, decision to publish, or preparation of the Wilschanski M, Pavlov V, Adileh M, Stojadi- manuscript. novic A, Avital I, Gure AO, Halle D and Nissan A. Colon Cancer Associated Transcript-1 (CCAT1) Disclosure of conflict of interest Expression in Adenocarcinoma of the Stom- ach. J Cancer 2015; 6: 105-10. None. [11] Ding C, Cheng S, Yang Z, Lv Z, Xiao H, Du C, Peng C, Xie H, Zhou L, Wu J and Zheng S. Long Address correspondence to: Drs. Gang Chen and non-coding RNA HOTAIR promotes cell migra- Zhenbo Feng, Department of Pathology, First Affi- tion and invasion via down-regulation of RNA liated Hospital of Guangxi Medical University, 6 binding motif protein 38 in hepatocellular car- Shuangyong Road, Nanning, Guangxi Zhuang Auto- cinoma cells. Int J Mol Sci 2014; 15: 4060-76.

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